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Aberrant cholesterol homeostasis is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease that is due to motor neuron (MN) death. Cellular toxicity from excess cholesterol is averted when it is enzymatically oxidized to oxysterols and bile acids (BAs) to promote its removal. In contrast, the auto oxidation of excess cholesterol is often detrimental to cellular survival. Although oxidized metabolites of cholesterol are altered in the blood and CSF of ALS patients, it is unknown if increased cholesterol oxidation occurs in the SC during ALS, and if exposure to oxidized cholesterol metabolites affects human MN viability. Here, we show that in the SOD1G93A mouse model of ALS that several oxysterols, BAs and auto oxidized sterols are increased in the lumbar SC, plasma, and feces during disease. Similar changes in cholesterol oxidation were found in the cervical SC of sporadic ALS patients. Notably, auto-oxidized sterols, but not oxysterols and BAs, were toxic to iPSC derived human MNs. Thus, increased cholesterol oxidation is a manifestation of ALS and non-regulated sterol oxidation likely contributes to MN death. Developing therapeutic approaches to restore cholesterol homeostasis in the SC may lead to a treatment for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNsin vitro. Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1G93Amice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1G93Amice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.SIGNIFICANCE STATEMENTNeurons that control muscular function progressively degenerate in patients with amyotrophic lateral sclerosis (ALS). Lipid dysregulation is a feature of ALS; however, it is unclear whether disrupted lipid homeostasis (i.e., lipid cacostasis) occurs proximal to degenerating neurons in the spinal cord, what causes it, and whether it contributes to neurodegeneration. Here we show that lipid cacostasis occurs in the spinal cord gray matter of ALS patients. Lipid accumulation was not associated with an aberrant increase in synthesis or reduced hydrolysis, as enzymatic and transcriptional regulators of lipid synthesis were downregulated during disease. Last, we demonstrated that genetic induction of lipid cacostasis in the CNS of normal mice was associated with ALS-like lipid pathology, astrogliosis, neurodegeneration, and clinical features of ALS.

Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T-α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T-SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T-SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.

Mutations in GBA1, the gene encoding glucocerebrosidase, are associated with an enhanced risk of developing synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. A higher prevalence and increased severity of motor and non-motor symptoms is observed in PD patients harboring mutant GBA1 alleles, suggesting a link between the gene or gene product and disease development. Interestingly, PD patients without mutations in GBA1 also exhibit lower levels of glucocerebrosidase activity in the central nervous system (CNS), implicating this lysosomal enzyme in disease pathogenesis. Here, we investigated whether modulation of glucocerebrosidase activity in murine models of synucleinopathy (expressing wild type Gba1) affected α-synuclein accumulation and behavioral phenotypes. Partial inhibition of glucocerebrosidase activity in PrP-A53T-SNCA mice using the covalent inhibitor conduritol-B-epoxide induced a profound increase in soluble α-synuclein in the CNS and exacerbated cognitive and motor deficits. Conversely, augmenting glucocerebrosidase activity in the Thy1-SNCA mouse model of PD delayed the progression of synucleinopathy. Adeno-associated virus-mediated expression of glucocerebrosidase in the Thy1-SNCA mouse striatum led to decrease in the levels of the proteinase K-resistant fraction of α-synuclein, amelioration of behavioral aberrations and protection from loss of striatal dopaminergic markers. These data indicate that increasing glucocerebrosidase activity can influence α-synuclein homeostasis, thereby reducing the progression of synucleinopathies. This study provides robust in vivo evidence that augmentation of CNS glucocerebrosidase activity is a potential therapeutic strategy for PD, regardless of the mutation status of GBA1.

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and β-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.